In this paper, we describe a pancreas cancer patient with an extraordinary disease course following treatment with chemotherapy and vaccination with autologous DC transfected with hTERT mRNA. In addition, we demonstrate how vaccination with full-length mRNA can be used for the discovery of novel, clinically validated T-cell epitopes.
The patient underwent radical surgery in January 2006. Due to post-operative complications, she did not receive adjuvant chemotherapy. The patient relapsed in November 2006 with enlarged lymph nodes localized in liver hilus, truncus iliacus and retroperitoneum as assessed by CT. The original diagnosis was confirmed by re-examination of the surgical specimen by an independent pathologist. The patient obtained stable disease on gemcitabine chemotherapy that was discontinued due to side effects. In this clinical setting, from June 2007, the patient has been vaccinated with autologous DC loaded with hTERT mRNA on a compassionate use basis in order to consolidate the beneficial effect of the chemotherapy.
Rather than developing progressive disease after ended chemotherapy, the patient experienced a partial response against the tumour. Two consecutive PET scans in March and September 2009 revealed no metabolically active lesions. These intriguing findings indicate that the immunotherapeutic strategy used has induced a clinically relevant immune response in this patient. It was therefore important to document and study in depth the immune response against hTERT. Furthermore, as there is a complete lack of information regarding the detailed immune response against hTERT from studies using full-length hTERT mRNA for vaccination [7
], it was important to identify hTERT epitopes potentially relevant in the antitumour response for the development of the next generation of hTERT vaccines.
We found high frequencies of CD8+ T-cell-binding pentamers with new CTL epitopes 674–683, but not 653–661 after vaccination. These two epitopes were HLA-A*0201 restricted and have not been previously described. Interestingly, the 9-mer epitope 674–683 is embedded in a 15-mer peptide 673–687 nearly identical to the R672 Th epitope previously described by Schroers et al. [14
]. Importantly, the same 15-mer peptide, 673–687, was also recognized by the T cells in proliferation assays, indicating that this region of the hTERT sequence may have elicited both a CD4+ and a CD8+ T-cell response in this patient. Moreover, seven other 15-mer peptides as well as one 30-mer peptide were recognized by T cells from this patient. Three of these peptides (663–677, 578–592 and 691–705) have not previously been reported. No immune responses against this set of peptides were observed when blood samples from six healthy donors were tested in the same assay (data not presented).
The amount of T-cell proliferation against these peptides was very variable. It is our general observation that immune responses against cancer vaccines in patients with concomitant tumours, as measured in peripheral blood, fluctuate considerably. This may reflect activated tumour-specific T cells leaving the blood stream to infiltrate the tumour. Stable immune responses are typically seen in patients where complete responses have been obtained. Induction of new hTERT-specific T cells by repeated booster vaccinations will contribute to the dynamic immune response. Taken together, these results demonstrate that the vaccine has induced T-cell responses against at least 10 different hTERT epitopes in this patient. The number of epitopes may be considerably greater as we have used only a limited number of peptides from an overlapping peptide library not covering the whole sequence as well as only a few pentamers limited to HLA-A*0201 presented peptides.
There is increasing emphasis on the pivotal role of CD4+ T cell in the anti-tumour response both in supporting CD8+ T-cell responses and for their direct anti-tumour effects [15
]. The observation of a CD4+ T-cell response against the 660–689 peptide, together with proliferative responses against several other peptides shown to be recognized by Th clones (Inderberg Suso et al., unpublished), in addition to CTL responses against several HLA-A*0201 restricted epitopes, supports the notion that a combination of T-helper (Th) and CTL epitopes should be used in future vaccination protocols. For this reason, the use of synthetic long peptides (SLP) in cancer vaccination is very promising. SLPs efficiently deliver antigen to DCs and are processed into both Th and CTL epitopes present in the peptide. The peptide presentation of SLPs which require internalization and intracellular processing has been shown to be prolonged compared to direct exogenous MHC class I molecule loading and presentation of short peptide not requiring processing [19
]. The prolonged peptide presentation is particularly important if the T-cell epitope displays weak MHC class I binding.
In addition, vaccination with short peptides can lead to tolerance of the immunizing antigens rather than immunity unless ex vivo loading of DC is used [20
]. Vaccinating with both CTL and Th epitopes has been shown to prevent this tolerance induction and the immunogenicity is further improved if these are included in one single hybrid peptide [22
]. This could provide an explanation for the strong immune responses and some of the extraordinary clinical responses seen in cancer patients vaccinated with GV1001, a 16-mer hTERT peptide [3
]. This Th epitope also incorporates HLA-A2- and HLA-B7-binding peptide motifs, and the strength of CD4+ T-cell responses detected against the GV1001 peptide correlates well with patient survival. Vaccination with full-length mRNA encoding defined antigens will also induce immune responses to both T-helper and CTL epitopes as demonstrated by the results obtained here.
Vaccination in advanced stage cancer patients will often have limited therapeutic effect by itself due to a combination of increasing immune suppression and rapidly growing tumour. The therapeutic effect may be enhanced when used in conjunction with conventional chemotherapy or radiotherapy, which may affect both regulatory T cells and tumour growth. Prior to the start of vaccination, the patient described here was treated with gemcitabine which may contribute to the induction of a broader immune response as shown by Nowak et al. [25
]. Furthermore, our observations indicate that there is no widespread tolerance against hTERT peptides and that the vaccination strategy using hTERT mRNA–transfected DCs is highly potent. Interestingly, despite the presence of a complex hTERT-specific immune response over a period of >3 years, no evidence of autoimmunity involving hTERT-positive normal cells, such as haematopoietic stem cells, was observed. This is in line with previous observations in patients with long-term immune responses following hTERT peptide vaccination [24
] and animal experiments [26
]. Our newly identified peptides add to the list of 26 hTERT peptides that have been shown to induce efficient immune responses against hTERT-positive tumour cells (reviewed in [27
We have also demonstrated that CD4+ T cells from this patient, capable of recognizing a 30-mer hTERT peptide (660–689) encompassing two of the 15-mer and one 9-mer peptide, produce three Th1-associated cytokines simultaneously. This kind of multifunctionality has previously been demonstrated to give better protection against infection [28
]. Peptide-specific cytokine secretion from CD8+ T cells could not be detected by flow cytometry and may reflect low initial precursor frequency of this CD8+ T-cell population. This difference in CD4+ and CD8+ T-cell numbers could be further increased by a much stronger proliferation of the CD4+ T cells recognizing the Th epitopes from the 30-mer peptide than of CD8+ T cells in the 12-day culture. The IFN-γ ELISPOT confirmed that cytokine secretion occurred mainly in response to Th epitopes.
In conclusion, these results illustrate the feasibility of vaccination with DCs loaded with mRNA encoding a defined antigen for the identification of immunogenic T-cell epitopes. The major advantage is that the identified epitopes have been processed and presented in vivo and therefore are potentially clinically relevant. In general, this type of vaccination provides an opportunity for direct and fast discovery of novel T-cell epitopes from any tumour-specific or tumour-associated antigen.